LOCUS       sequence-490908-        6633 bp    DNA     circular SYN 28-OCT-2025
DEFINITION  All-in-one plasmid. Expresses R-IscB_ADAR2dd in mammalian cells for 
            A-to-I RNA editing. Inactive mCherry included to assess A-to-I 
            editing. Spacer included can direct mCherry fluorescence recovery..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6633)
  AUTHORS   Xu C, Niu X, Sun H, Yan H, Tang W, Ke A
  TITLE     Conversion of IscB and Cas9 into RNA-guided RNA editors.
  JOURNAL   Cell. 2025 Aug 18:S0092-8674(25)00854-2. doi: 
            10.1016/j.cell.2025.07.032.
  PUBMED    40829585
REFERENCE   2  (bases 1 to 6633)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 28, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2025 
            Aug 18:S0092-8674(25)00854-2. doi: 10.1016/j.cell.2025.07.032."
COMMENT     Sequence Label: pMini-CMV-NLS-dead R-IscB_ADAR2dd-NES_T2A_mChe...
FEATURES             Location/Qualifiers
     source          1..6633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     polyA_signal    510..565
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(564..583)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    594..818
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     807..824
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(856..1566)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFK*ERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKARKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1134..1153)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1403..1421
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1567..1620)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(1627..1653)
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             complement(4246..4266)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4269..4278
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4303..4506)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4332..4352)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4507..4886
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4894..4998
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4999..5067,5068..5859)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5217..5236)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6030..6618
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6519..6538
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg tcctgtcctc gaagttcatc acgcgctccc acttgaagcc ctcggggaag
      301 gacagcttca agtagtcggg gggctctttc acttgaaaag gttgaaagag cacaggctga
      361 gacattcgta aggccgaaag gccggacgca ccctgggatt tccccagtcc ccggaactgc
      421 atagcggatg tcagttgatg aaaatcagat aagccagggg gaacaatcac ctctctggaa
      481 acagagagag ttttttttgt tttgcagtaa ataaaggaaa tttattttca ttgcaatagt
      541 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcccataga
      601 gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg
      661 ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc
      721 attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga
      781 ggggcaaaca acagatggct ggcaactaga aggcacagtc gaggctgatc agcgggttta
      841 aacgggccct ctagattact tgtacagctc gtccatgccg ccggtggagt ggcggccctc
      901 ggcgcgttcg tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt
      961 gatgttgacg ttgtaggcgc cgggcagctg cacgggcttc ctggccttgt aggtggtctt
     1021 gacctcagcg tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt
     1081 cagggcgccg tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt
     1141 ctgcattacg gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa
     1201 ctcgccgtcc tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt
     1261 catcacgcgc tcctacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc
     1321 ggcggggtgc ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca
     1381 ggcgaagggc agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta
     1441 ggggcggccc tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat
     1501 gtgcaccttg aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct
     1561 caccattggg ccgggatttt cctccacgtc cccgcatgtt agaagacttc ccctgccctc
     1621 ggatcccagt gtcagtcttt caagtggagg cagttgaagg gatcccgtga gtgagaactg
     1681 gtcctgctcg gtgggcttct ccacccaggc ccccagcccc gccttgatga aggctgtgaa
     1741 cagacgcgcc ttggcggcct ggtactcctt tgccgccagc ttggactcat ggtacacgtt
     1801 gggcttggta atcttggagc gtagtaagtg ggagggaacc ttgccgtgca cacgcatcca
     1861 gcgacagtac aacgcgtgct tacacaggcg ggacgcgcgg cccagctcat ccttcccagt
     1921 cgtggcgttg atgacctcaa tagcggagtc gcctaccgtc cagttgacac tgaagttggg
     1981 ggccttccct ggctgccgtg cttctgcatt gctgatgcca ctgagcaaag gcttgttgag
     2041 ggtgtagaga ggtggcaggt cctctatgtt ggagatccgc tggtacatgg ccctggaaag
     2101 gtggtccccg tggtaaaggc tgcccaggat gatgctcgag aagtaaatgg gctccacgaa
     2161 aatgctgagc agtgatccct ggatgcccac cacgttccag cgtgcaatct tgtcactgca
     2221 ggacatggtg agcagccgct ccccttgcag caccccgtcc cacgtttgga tgctcgcatt
     2281 ggagcgcact ggaatcgtcc cctgaccaga ctctattttg gtccgtagct gtcctcttgc
     2341 tttacgattt gggtgtctat ctgctggttc ttccaggatt ggctcatgtg gtgagaagat
     2401 tctggcatct ccacagggag aggtgctgat gtacagatga aactggacat tctccttcag
     2461 cctaaacccc cctcgctctg atttctgaaa gatggatctt ttttgatcat ctttgttatt
     2521 taagtaaagc tcaagttgtg tataaagaaa tctgagcaag gatctccgag atattatttc
     2581 tgcatggcag tcatttaatg caaggccacg atcactcatg tattcaccat taatacattt
     2641 gcctcctgta gaaacactta tcaccttggc atctttaaca tctgtgcctg ttgtcatgac
     2701 gactccagcc agcacttttc tgcgagcgtg aggggaggag aagttgtcgg tcaggtcacc
     2761 aaacttaccc aggaccaggc gtgagacagc gtcagctaaa acctgcggta aatgcagctg
     2821 tgaccccccg ctgctgcccc cgctgctttc aggtgttgcg ctctcgcttg tgccaggtgt
     2881 ctcagagcca gaggagcctc cgctagatcc tccggacacg tagatctgca gtccgccgtt
     2941 gtactgagca gaagggtgct tcacggtcag tttagacacg tcagcggcgc tgtgagcggc
     3001 tctgtactct tccaggctgt cagttttctg gtccatagct ttgtgccggt tggtggcaac
     3061 tttcttgccg cccatgtagt agctccggtt cagattggcc ttgtgacagg cctgtctatc
     3121 atgccttctg aactggtgca ccatgtatgg tctgccctta gggctgctca ccttcttggc
     3181 gtcggtcagt gcgctgcagg cgatgcagta ggcgtccagg tagtggtcct tagggatgcc
     3241 atgctcttcc cgaaacagat aggtgtcttg gccgcttgtc acgcagaaat ttccggggaa
     3301 catatcggcc agctgatcag ccaggtaggg gatgatctgg ttcagcacag acagggcgtg
     3361 gtatttcttg ttcattccag acttcttgct ggccaggttg gcctcccact ccttatctgt
     3421 gtgcaccagt ctcgcgtgtt cttcacaaag gcccactctg ttttccagtg tctcgctgcc
     3481 gttctttctt ctggggacca cgtggtggta gtggtcgatg ccgtgtttac aaaacaggca
     3541 gtgtccatcc tgctgcatgg acacggcctc ttccacgctg cctttgccgt acagagggcc
     3601 ccgctggtac tgccatctct gaaccttggg gttgttcatg gccataaagc tgaaccgatt
     3661 cagctccagc accaccttgg ccacgggcag gatcttctgc accttcttaa ccacgttcag
     3721 gtgggtgacc agcaggtgat tggcggtagg tgtcagccat cctttaggcc gcttgcggtt
     3781 gttgaatctg gcttccttgt tccggatgct cttgcatgta ataggctttt tgcagccagg
     3841 cagcagtctt tgcttttcgc cctcctcgaa ggcggtgccg gcagccttgg ctctccgccg
     3901 tcttttgcac cgccgcttca gccttctgtg ggcacgccta tactgctttc tgtccttcat
     3961 cagcttaggc acgtccttgt ttcttgtctc gatttgggcg ttgaacacgc tctctccgct
     4021 ctcggtcacc acgctcatac cgatattggt tctgccagga gcaatgccca gcaccagagg
     4081 ctgggtttcc tcggcagact cgtaggtcag ctggatggtg aagggctttc tttccaccac
     4141 tctagccttg ccttccttga gcaagatccg cacgtggccg catctggtgg tgggcatcag
     4201 tggtttgccg gacttagaga tcacgtagac cacggccata gatcccacct tccgtttctt
     4261 ttttggtccc atggtggcag tgggttctct agttagccag agagctctgc ttatatagac
     4321 ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt
     4381 tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag
     4441 acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg
     4501 catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca
     4561 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     4621 gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     4681 ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat
     4741 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     4801 ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta
     4861 ttaataacta gtcaataatc aatgtcaaat gtgcgcggaa cccctatttg tttatttttc
     4921 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     4981 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5041 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5101 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5161 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5221 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5281 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5341 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5401 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5461 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5521 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5581 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5641 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5701 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5761 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     5821 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     5881 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     5941 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6001 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6061 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6121 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6181 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6241 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6301 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6361 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6421 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6481 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6541 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6601 gggcggagcc tatggaaagg catgcaagct tgg
//
